Materials
Reagent: Chloral hydrate (8MQ20-CC) was purchased from TCI Shanghai; Western and IP cell lysate (P0013) was purchased from Beyotime Biotechnology; Protease inhibitor cocktail (without EDTA, 100× DMSO storage solution) (B14002) and phosphatase inhibitor cocktail (100 × B15002) were purchased from Bimake; Rat IL-1β ELISA Kit (GN-R30172), rat IL-18 ELISA Kit (GN-R30168), rat TNF-α ELISA Kit (GN-R31092) and rat IL-6 ELISA Kit (GN-R30201) were purchased from Gaining Biological; LPS detection kit (H178) was purchased from NanJing JianCheng Bioengineering Institute; GSDMD mouse monoclonal antibody (SC-393581) and caspase-11 (SC-374615) were purchased from Santa Cruz Biotechnology; GSDMD-N rabbit monoclonal antibody (93709) was purchased from Cell Signaling Technology; Caspase-1 (NBP1-45433), NLRP3 (NBP2-12446) Rabbit anti polyclonal antibodies were purchased from Novus Biologicals; ASC (EPR10402(B))Rabbit monoclonal antibody was purchased from abcam; β-actin mouse anti monoclonal antibody (A5316) was purchased from Sigma; Goat anti rabbit second antibody (AP132P) and Goat anti mouse second antibody (AP124P) were purchased from Merck.
Instrument: Microplate Reader, Electrophoresis apparatus, Film transfer apparatus and Gel imaging system (BIO-RAD, USA), LEICADNLB2 binocular microscope (LEICA company), Shandon325 paraffin slicing machine (British Shandon company), Fall-automatic biochemical analyzer.
Preparation of Jiangzhi Ligan Decoction
JZLGD is composed of Alisma orientalis(Zexie) 10 g, Cassia seed(Juemingzi)30 g, Salvia miltiorrhiza(Danshen) 10 g, Turmeric (Yujin) 10 g, Seaweed(Haizao)30 g and Lotus leaf (Heye)10 g. All the Chinese herbal pieces were purchased from The First Hospital of Hunan University of Chinese Medicine. The above drugs were soaked in 8 times of water for 30 min, boiled for 1 h, then filtered, added 6 times of water, boiled for 1 h, and then filtered. We mixed the liquid medicine and concentrated it to 2g /ml by using Rotary Evaporator at 65℃, and stored it at 4℃. Before use, it was diluted to the required concentration with normal saline.
Animals and treatment
Sprague-Dawley(SD) male rats (150g ± 10g) were purchased from Hunan SJA Laboratory Animal Co.,Ltd and acclimatized for 7days. Rats were kept in controlled ambient temperature (24 ± 2 °C)and humidity (60 ± 10%) under 12 h light-dark cycles; water was available ad libitum. This study was reviewed and approved by the Animal Experiment Ethics Committee of Hunan University of Chinese Medicine and carried out in accordance with their recommendations.
According to the previous study, the rats received a high-fat diet (HFD) (40 kcal% fat, 20 kcal% sucrose, and 2% cholesterol) for 12 weeks to develop NAFLD. The high-fat diet was provided by
SD rats were randomly divided into five groups: normal control group, NAFLD model group and JZLGD-treated NAFLD group (3 doses of JZLGD: 2.3, 4.6, 9.2 g/kg of body weight, respectively). The normal control group were fed a normal diet(ND), while the rest of the groups received a HFD during the experimentation period (12 weeks + 6 weeks). After 12 weeks, rats in JZLGD-treated NAFLD group were administered different doses of JZLGD by oral gavage once daily for another period of 6 weeks, and the rest were given the same dosage of normal saline. Food intake was monitored daily and body weight was measured weekly during the experiment. After the intervention, blood and liver from each sample were rapidly removed and stored at -80℃ for further studies.
Liver index
After 18 weeks , the rats were anesthetized by injecting with 1% pentobarbital sodium after overnight fasting. The body weight and liver weight were measured, and the following formula was used for determining the liver index: liver index = (liver weight/body weight) * 100%.
Histopathology analysis
For histopathological analysis, we took the same segments of liver tissue from each group and washed it with ice-cold saline for 1-2 times, then kept them in 4 % paraformaldehyde. 4μm thickness tissue were embedded in paraffin blocks and stained with hematoxylin-eosin. Three practiced pathologists who were blinded to the study design performed all the histopathological examination, then scored the ballooning degeneration score and the NAFLD activity score. The scoring criteria were in accordance with the guidelines of the National Institutes of health clinical research network on non-alcoholic steatohepatitis.
Biochemical estimations
The blood samples were centrifuged at 3000 r/min for 10 min to separate the serum. We used fall-automatic biochemical analyzer to evaluate serum activity of liver enzymes (alanine transaminase enzyme (ALT) and aspartate transaminase enzyme (AST)) and serum level of lipid profile (triglyceride(TG), total cholesterol(TC), low density lipoprotein(LDL) and free fatty acids(FFA)).
Assessment of serum IL-1β, L-18, TNF-α, IL-6
Blood was obtained from abdominal aorta and centrifuged to get the serum. The levels of IL-1β, L-18, tumor necrosis factor α(TNF-α) and IL-6 in serum were determined by enzyme-linked immunosorbent assay technique (ELISA) according to standard instructions.
The levels of serum LPS
Blood was obtained from the abdominal vein and placed at room temperature for 30 minutes, then centrifuged to separate the serum. The level of serum LPS was measured with a kit according to the manufacturer’s instructions. Firstly, samples were added into the enzyme labeled wells which had been coated with antibodies, and then labeled recognition antigens were added. The mixture was allowed to incubate at room temperature for 1 h. Afterwards, the plate was washed with PBST three times, then we added avid in HRP and made them incubated at 37 ℃ for 1 h. The absorbance was measured at 450 nm. The standard curve was drawn according to the absorbance of standard pore and its corresponding concentration, and the concentration of LPS in serum of each group could be calculated.
Western blot analysis
We randomly selected five mice in each group, and took the same segments of liver tissue 100 mg. Then 1 ml RIPA (with cocktail and phosphatase inhibitor added) was added. After homogenize on ice with a glass homogenizer for 30 min, the homogenate was centrifuged to obtain the hepatic tissue proteins, the concentration of protein extracted was determined with BCA. We took 40 ug protein in each group for the follow-up experiment. Protein samples were separated by SDS-PAGE gels and transferred to PVDF membranes. After blocking with 5 % nonfat milk in TBST for 1 h, membranes were incubated at 4 °C overnight with the primary antibodies against NLRP3 (1:1000), apoptosis-associated speck-like protein containing a CARD(ASC)(1:1500), Caspase-1 (1:500), Caspase-11 (1:500), GSDMD (1:1000), GSDMD N-terminus(GSDMD-N) (1:1000), IL-1β (1:1000), IL-18 (1:1000) and β-actin (1:5000). After washing the membrane with TBST, the membranes were incubated at room temperature with secondary antibodies for 1 h. The protein bands were visualized with Western Bright ECL. Finally, Quantity One software was used for the quantitative grayscale analysis of the band
Statistical analysis
The grouped data were analyzed with SPSS 22.0. Comparison of two groups was performed by two tailed Student's t-test. Comparison among three or more groups were analyzed with one-way ANOVA test. Results were expressed as the mean ± SD(standard deviation). The GraphPad Prism6.0 software was used for statistical evaluations. P<0.05 was considered to indicate a statistically significant difference.